Purpose: We aimed to evaluate the effect of long-term oral OM-89 therapy on the urinary microflora in patients with urolithiasis. Materials and Methods: Patients underwent surgical removal of urinary stones followed by no OM-89 treatment for six months or daily OM-89 administration. Urine culture and urinary polymerase chain reaction (PCR) were performed at the baseline visit (V1) and at 2 months (V2) and 6 months (V3) after the operation. Results: A total of 113 patients completed the study. The rate of urinary bacteria detection by urine culture at V3 did not differ between OM-89 treated and untreated groups (p>0.999); however, the PCR detection rate tended to be higher in OM-89 untreated group than in OM-89 treated group (p=0.052). Escherichia coli and Enterococcus spp. were the bacteria most commonly detected via both urine culture and PCR at all timepoints. Risk factors for the detection of bacteria by urine culture at V3 were positive culture at V1 (p=0.048) and female sex (p=0.048), whereas positive PCR at V3 was associated with female sex (p=0.023), positive PCR at V2 (p<0.001), and no OM-89 treatment (p=0.038). The use of OM-89 was associated with decreased rates of bacterial detection by PCR at V2 and a further decrease at V3. Conclusions Long-term immunization with OM-89 could further decrease the frequency of urinary bacterial colonization after surgical removal of urinary stones. OM-89 could be used as a complementary therapy if a retrieved stone is suspected to be related to infection.